| Objective:Hepcidin is a peptide hormone synthesized and secreted mainly by the liver,which is the main regulator of iron in the body[1].The position-64/-72 in the Hepcidin promoter is a STAT3 binding motif,which can regulate the synthesis of Hepcidin by Janus kinase-signal transducer and activator of transcription 3(STAT3)signaling pathway,under inflammatory condition especially when IL-6 is high.Increased Hepcidin can lead to chronic diseases such as chronic inflammation,infection and cancer by reducing iron absorption,increasing intracellular iron storage,and promoting cell survival [2].Elevated Hepcidin levels in serum and tissues of cancer patients are generally considered to be an indirect consequence of increased levels of cytokines and the stimulatory effects of cytokines on Hepcidin synthesis[3].Dysregulation of Hepcidin expression may not only alter the systemic regulation of iron,but also have local effects on tumor growth and malignant transformation.Hepcidin plays an important role in iron homeostasis,and the occurrence and development of tumor[4].Hepcidin binds to ferroportin which is an iron efflux pump expressed in enterocytes and macrophages of the reticuloendothelial system.The compound of hepcidin-ferroportin triggers ferroportin degradation,thereby blocking iron delivery from enterocytes to the systemic circulation,as well as blocking delivery of iron which is catabolized by macrophages to the circulation[5].Iron plays an important role in the development of tumor through Hepcidin or regulating the protein of Hepcidin expression,including inflammatory mediators or FPN,which paves the way for the targeted therapy of iron and Hepcidin [6].Therefore,this study is to investigate whether the Hepcidin level in gastric cancer is increased,whether the IL-6-STAT3 signaling pathway is involved in the regulation of Hepcidin,and to explore the effect of Hepcidin on the biological behavior of gastric cancer and its mechanism.The objective is to determine the expression level of Hepcidin in gastric cancer and its regulatory pathway,the effect of Hepcidin on the proliferation and metastasis of gastric cancer cells and its mechanism,and to find a new target for the treatment of gastric cancer.The first chapter: IL-6-STAT3 signaling pathway regulates Hepcidin levels Methods:1.Apply double antibody sandwich enzyme-linked immunosorbent assay to detect the level of Hepcidin and interleukin-6 in serum of gastric cancer patients,and statistically analyse the relationship between Hepcidin and IL-6.2.Apply real time fluorescent quantitative PCR to detect m RNA Level of Hepcidin in gastric tissue,Apply Western Blot to detect the expression of Hepcidin protein and p STAT3 protein which is the downstream signal molecules of IL-6,and statistically analyse the relationship between Hepcidin and clinicopathological characteristics,tumor metastasis gene Tiam1 and Rac1,the relationship between Hepcidin protein and p SATA3 protein.3.Using exogenous IL-6 to treat gastric cancer cells,apply real time fluorescent quantitative PCR and Western Blot to detect the expression level changes of Hepcidin m RNA and Hepcidin protein and p STAT3 protein in gastric cancer cell lines,apply the immunofluorescence test to detect Hepcidin positioning,confirm that the IL-6-STAT3 signaling pathway is involved in the regulation of Hepcidin levels in gastric cancer cell.Results:1.The level of serum Hepcidin in gastric cancer group was 10.38(7.10,19.35)ng/ml,higher than that in benign gastric disease group 7.43(1.88,12.24)ng/ml and normal control group 7.40(4.08,12.05)ng/ml,there was statistically significant differences(P<0.05),while there was no significant difference between benign gastric disease group and normal control group(P>0.05).The level of serum IL-6 in gastric cancer group was 3.16(1.69,4.87)pg/m L,higher than that in normal control group 0.87(0.56,0.99)pg/m L,there was statistically significant differences(P<0.05),lower than that in benign gastric disease group 4.17(2.36,5.27),while there was no significant difference(P>0.05).There was a positive relationship between level of serum Hepcidin and IL-6 in gastric cancer group(r=0.459,P=0.000).2.The positive expression rates of Hepcidin m RNA in gastric cancer group tissues and gastric benign disease group tissues were 61.54%(40/65)and 58.82%(10/17)respectively,higher than that in gastric normal group tissues,there were statistically significant differences(P<0.05);The level of Hepcidin m RNA in gastric cancer group tissues was-0.38±1.05,higher than benign gastric disease group tissues-0.51±0.65 and gastric normal group tissues-1.65±0.89,there were statistically significant differences(P<0.05).The positive expression rates of Hepcidin protein in gastric cancer group tissues in gastric cancer group tissues and gastric benign disease group tissues were 60%(39/65)and 58.82%(10/17)respectively,higher than that in gastric normal tissue 20%(4/20),there were statistically significant differences(P<0.05).The positive expression rates of p STAT3 protein in gastric cancer tissues in gastric cancer group tissues and gastric benign disease group tissues were 46.15%(30/65)and 47.06%(8/17)respectively,higher than that in gastric normal group tissue 0(0/20),there were statistically significant differences(P<0.05).There was positive correlation between Hepcidin protein and p STAT3 protein.3.With the increase of exogenous IL-6 concentration,the expression of Hepcidin m RNA and protein and p STAT3 protein in gastric cancer cell SGC7901 increased,and the expression level of Hepcidin m RNA was highest at IL-6 concentration of 20ng/ml.Cell immunofluorescence assay showed that Hepcidin protein was increased in gastric cancer cell MGC803,and was mainly concentrated in the cytoplasm and near the cell membrane.IL-6-STAT3 signaling pathway is involved in the regulation of Hepcidin.The second chapter: The levels of Hepcidin influence proliferation and metastasis of gastric cancer cell Methods:1.To analyze the relationship between Hepcidin in serum and tissues of gastric cancer and clinicopathological features and tumor metastasis gene Tiam1.2.Using exogenous Hepcidin to treat gastric mucosal epithelial cell and gastric cancer cell,apply MTT and cell scratch healing test to detect the effects of exogenous Hepcidn on the proliferation of gastric mucosal epithelial cell and metastasis of gastric cancer cells.3.Constructing and transfecting eukaryotic expression vector of Hepcidin to increase the expression levels of Hepcidin and RNA interference technology to reduce the expression level of Hepcidin,apply flow cytometry assay and cell scratch healing test to detect the effect of increased and decreased Hepcidin on proliferation and metastasis of gastric cancer cells.Results:1.Statistically analyse according to the depth of tumor invasion depth,lymph node metastasis,distant metastasis and clinical stage of TNM,the level of Hepcidin in T3+T4 gastric cancer group serum was 11.26(8.63,23.77)ng/m L,higher than that in T1+T2 8.78(3.66,11.25)ng/m L,the difference was statistically significant(P=0.017);The level of Hepcidin in N1-3 gastric cancer group serum is 10.73(7.90,22.35)ng/m L,higher than N0 10.18(5.31,12.78)ng/m L,but the difference was not statistically significant(P=0.280);The level of Hepcidin in M1 gastric cancer group serum was 10.72(8.60,30.85)ng/m L,higher than M0 10.36(4.31,18.48)ng/m L,but the difference was not statistically significant(P=0.215);The level of Hepcidin in III + IV gastric cancer serum was 11.21(8.56,20.60)ng/m L,higher than that in I + II 9.31(3.83,17.01)ng/m L,but the difference was not statistically significant(P=0.240);The level of Hepcidin in cancer group serum was related with tumor invasion depth,but not with lymph node metastasis,distant metastasis and clinical stage of TNM.The level of Hepcidin m RNA in T3+T4 gastric cancer group tissues was-0.23±0.84,higher than that in T1+T2-0.74±0.50,the difference was statistically significant(P=0.038);The m RNA level of Hepcidin in N1-3 gastric cancer group tissues was-0.39±0.81,higher than that of N0-0.66±0.55,the difference was statistically significant(P=0.046);The level of Hepcidin m RNA in M1 gastric cancer group tissues was-0.25±0.70,higher than that in M0-0.65±0.50,the difference was statistically significant(P=0.043);The level of Hepcidin m RNA in III + IV gastric cancer group tissues was-0.23± 0.75,higher than that in I + II-0.63±0.47,the difference was statistically significant(P=0.030).The m RNA levels of Hepcidin in gastric cancer group tissues was positively related to tumor invasion depth,lymph node metastasis,distant metastasis and clinical stage of TNM.There was a positive correlation between the levels of Hepcidin m RNA and Tiam1 m RNA in gastric cancer group tissues(r=0.908,P<0.05).The level of IL-6 in T3+T4 gastric cancer group serum was 2.51(1.74,4.72)ng/m L,lower than that in T1+T2 3.25(1.60,5.25)ng/m L,but the difference was not statistically significant(P=0.561);the level of IL-6 in N1-3 gastric cancer group serum was 2.75(1.73,4.57)ng/m L,lower than that in N0 3.27(1.50,5.69)ng/m L,but the difference was not statistically significant(P=0.674);the level of IL-6 in M1 gastric cancer group serum was 3.46(2.05,4.91)ng/m L,higher than that in M0 2.80(1.55,4.87)ng/m L,but the difference was not statistically significant(P=0.597);The level of IL-6 in III + IV gastric cancer serum was 3.38(1.86,4.98)ng/m L,higher than that in I + II 2.75(1.59,4.37)ng/m L,but the difference was not statistically significant(P=0.575).2.With exogenous Hepcidin to treat gastric mucosal epithelial cell GES1 and gastric cancer cell line SGC7901,the result of MTT experiment show that with the increase of Hepcidin concentration,the absorbance(OD)increased,suggesting that the proliferation of gastric mucosal epithelial cell GES1 enhanced,the result of cell wound healing experiment show that with the increase of Hepcidin concentration,cell grow from both sides of scratch to the central,suggesting that the migration capacity of gastric cancer cell line SGC7901 enhanced.3.The m RNA level of Hepcidin in gastric cancer cell line SGC7901 with Hepcidin eukaryotic expression vector was higher than that in SGC7901 with empty vector,the difference was statistically significant(P<0.05),the expression level of Hepcidin protein increased,suggesting that the Hepcidin eukaryotic expression vector was transfected into SGC7901 and stably expressed Hepcidin protein;The results of flow cytometry showed that the G0/G1 phase of SGC7901 in experimental group was 47.06%,lower than that in control group 57.32%,S2 and G2 were 52.94%,higher than that in control group 42.68%,suggesting that elevated Hepcidin can promote proliferation and growth of SGC7901;The results of wound healing test show that cells grow from both sides of scratch to the central,scratch distance is narrow,suggesting that elevated Hepcidin can promote migration of SGC7901.4.The m RNA level of Hepcidin in gastric cancer cell line MGC803 with Hepcidin silencing by si RNA was lower than that in control group,the difference was statistically significant(P<0.05),Hepcidin protein expression decreased,suggesting that si RNA silenced and decreased Hepcidin expression.The results of flow cytometry showed that G0/G1 phase of MGC803 cells in the experimental group was 46.12%,higher than that in the control group 38.10%,S2 and G2 was 53.88%,lower than that in the control group 62.90%,the expression level of Hepcidin decreased in gastric cancer cell line MGC803,suggesting that decreased Hepcidin can inhibit the proliferation and growth of MGC803.The results of wound healing test showed the gastric cancer cells with decreaed hepcidin cannot grow from both sides of cell scratch to the central growth,scratch distance is not narrow,suggesting that reduced Hepcidin has obvious inhibiting effect on cell migration.The third chapter: Hepcidin-Ferroportin signaling pathway promts proliferation and metastasis of gastric cancer cell Methods:1.Apply real time fluorescence quantitative PCR to detect the m RNA expression level of FPN in gastric cancer tissue and gastric cancer cells,and statistically analyse the relationship between FPN and clinicopathological features,the relationship between Hepcidin and FPN.2.Appy real time fluorescent quantitative PCR and bathophenanthroline method to detect the changes of FPN and available iron pool level in gastric cancer cell line after exogenous Hepcidin treatment,confirm that Hepcidin-FPN-Iron signaling pathway is invoveld in proliferation and metastasis of gastric cancer cell.Results:1.The m RNA expression positive rates of FPN in the gastric cancer group and the gastric benign lesion group were 30.77%(25/65)and 29.41%(5/17)respectively,lower than that in the gastric normal control group 75%(15/20),and the difference were statistically significant(P<0.05).The m RNA level of FPN in the gastric cancer group was-0.54±0.51,which was lower than that in the gastric benign lesion group-0.31±0.58 and in the normal gastric control group-0.15±0.60,and the difference were statistically significant(P<0.05).There was a negative correlation between the level of FPN m RNA and Hepcidin m RNA in the gastric cancer group tissues(r=-0.747,P=0.000).The level of FPN m RNA in T3+T4 gastric cancer group tissues was-0.54±0.36,lower than T1+T2-0.21±0.66,the difference was statistically significant(P=0.035);The level of FPN m RNA in N1-3 gastric cancer group tissues was-0.56±0.66,Lower than that in N0-0.26±0.56,the difference was statistically significant(P=0.034);The level of FPN m RNA in M1 gastric cancer group tissues was-0.55±0.42,Lower than that in M0-0.27±0.48,the difference was statistically significant(P=0.043);The level of FPN m RNA in III + IV gastric cancer group tissues was-0.56±0.32 Lower than that in I + II-0.29±0.41,the difference was statistically significant(P=0.045);The level of FPN m RNA in gastric cancer group tissues was negatively correlated with tumor invasion depth,lymph node metastasis,distant metastasis and clinical stage of TNM.The expression of FPN in gastric cancer cells with high expression of Hepcidin decreased;on the contrary,the expression of FPN in gastric cancer cells with low expression of Hepcidin elevated,and the expression of FPN in gastric cancer cells was negatively correlated with the expression of Hepcidin.2.The expression of FPN was decreased and the level of available iron pool was increased in cells treated with exogenous Hepcidin,Hepcidin-FPN-Iron involved in the proliferation and metastasis of gastric cancer cells.Conclusions:1.Through the detection of Hepcidin and IL-6 in serums,Hepcidin and p STAT3 in tissues,the level of Hepcidn,IL-6,Hepcidn m RNA,Hepcidin protein and p STAT3 protein were increased,the level of IL-6 and Hepcidin,the expression of Hepcidin protein and p STAT3 protein were positively correlated;Through the detection of changes of p STAT3 protein,Hepcidin m RNA and Hepcidin protein in gastric cancer cell treated by IL-6,when the level of IL-6 increased,the expression level of p STAT3 protein increased,thus the level of Hepcidin increased,demonstrated that IL-6-STAT3 signaling can positively regulate the level of Hepcidin in gastric cancer.2.Through analyzing the relationship between Hepcidin and clinicopathological features in gastric cancer group,Hepcidn and the depth of tumor invasion,lymph node metastasis,distant metastasis and TNM staging were positively correlated.Further by the addition of exogenous Hepcidin,transfecting expression plasmid to increase the expression of Hepcidin in cells,transfecting si RNA to decrease expression of Hepcidin in cells,changes using MTT method,flow cytometry assay and cell scratch assay to detect the change of cell proliferation,cell cycle and cell migration,confirmed the elevated level of Hepcidin can promote proliferation and metastasis of gastric cancer cell,the reduced level of Hepcidin can inhibit proliferation and metastasis of gastric cancer cell.3.Through the detection of FPN and Hepcidin of the tissues and cells,the level of serum iron,the detection of changes of FPN and available iron pool level detection gastric cancer cell lines treated by exogenous Hepcidin,Hepcidin binding to ferroportin can trigger the internalization of FPN and increase intracellular iron to promote the proliferation and metastasis of gastric cancer cells,confirmed Hepcidin-FPN-Iron play an important role in malignant transformation. |
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